CN102090338A - Rapid tissue culture propagation method for miscanthus floridulu somatic embryo of mscanthus plant - Google Patents
Rapid tissue culture propagation method for miscanthus floridulu somatic embryo of mscanthus plant Download PDFInfo
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Abstract
The invention discloses a rapid tissue culture propagation method for miscanthus floridulu somatic embryo of mscanthus plant, comprising the following steps: A, selecting young ear: selecting the yound ear at Ying flower primordium formation period; B, carrying out primary induction with a primary culture medium: cutting to small sections, sterilizing with alcohol, soaking with mercuric chloride, washing and inoculating on the primary induction culture medium; C, subculturing seedling with a seedling culture medium: subculturing and generating the seedling on a subculture culture medium with the obtained embryonic callus, subculturing, multiplication and shining every day; D, carrying out radication induction with a radication induction culture medium; carrying out the induction radication on the radication induction culture medium by seedling, culturing and shining every day; E, acclimating and transplanting: transplanting the tissue culture seeding to an acclimation nursery garden consisting of vermiculite to acclimate, spraying acclimation culture solution and clean liquid to the seeding at fixed time every day, transplanting to the nursery garden and obtaining the miscanthus floridulu seeding. The method is easy to apply, simple to operate, and large in breed coefficient; and the recovery reproduction rate is high, which is about 100%.
Description
Technical field
The present invention relates to Caulis Miscanthis floriduli (
Miscanthus floridulu) based on the group culturation rapid propagating technology field of embryo callus subculture, more specifically relate to a kind of awns platymiscium Caulis Miscanthis floriduli body embryo tissue culture quick propagation culturing method, it is adapted to Caulis Miscanthis floriduli children fringe is that explant passes through callus of induce approach acquisition seedling, and rises in value, takes root, tames and transplant.
Background technology
Domestic group culturation rapid propagating technology about the awns platymiscium, relevant report was just arranged in the nineties in 20th century, they have adopted spire, hypocotyl, young root respectively, children's fringe carries out the fast breeding technique of awns platymiscium as explant, but its healing rate and differentiation rate all have nothing in common with each other, reproductive efficiency with young fringe is the highest, but does not obtain embryo callus subculture.In addition, also relevant for the report of awns platymiscium group culturation rapid propagating technology, utilized stem stalk cuttage technique to obtain seedling, but this also is the adventitious organogenesis that utilizes, and does not obtain embryo callus subculture in 2005.And our research is to utilize young fringe to obtain embryo callus subculture for explant, continues to do experiments such as differentiation, transplanting again.Therefore can directly do relevant subsequent tests such as transgenosis, not only reach purpose rapidly and efficiently, the transgeneic procedure for Caulis Miscanthis floriduli provides reliable group of training system simultaneously.
Summary of the invention
The objective of the invention is to be to provide a kind of awns platymiscium Caulis Miscanthis floriduli body embryo tissue culture quick propagation culturing method, easy to implement the method, easy and simple to handle, reproduction coefficient is big, and the callus reproduction rate height of acquisition is about 100%.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
China is the center of awns platymiscium resource distribution, and the distribution of Caulis Miscanthis floriduli is wider.Unmatched barren place growths such as Caulis Miscanthis floriduli can be in the deserted mountain, barren hill, hills, rivers lakeshore, it is extremely well-adapted not only, and can solve can the grain battle contradiction.The growth cycle of Caulis Miscanthis floriduli is short, and generally its growth cycle is about 180 days, and photosynthesis is relative with photomorphogenesis shorter, does not influence the structure of the ecosystem after the results, and biomass is big, and is flammable strong, can realize CO during as energy resource consumption
2Zero-emission.In view of energy crisis in the present worldwide and the research of domestic less related science, researcher should make full use of the herbaceous biomass resource of this class high-quality of China, this class plant is entered the research in a dark step.Content of the present invention comprises that with Caulis Miscanthis floriduli children fringe be the initial culture base of explant induction embryo callus subculture, gives birth to the seedling subculture medium, and the root induction medium of seedling.
The present invention sets up the Caulis Miscanthis floriduli tissue-culturing rapid propagation system based on embryo callus subculture, appreciation rate height (5-6 is doubly), and increment is stable, the root system stalwartness, the transplanting survival rate height is to be suitable for the commercial mature technology that expands numerous Caulis Miscanthis floriduli.
A kind of awns platymiscium Caulis Miscanthis floriduli body embryo tissue culture quick propagation culturing method the steps include:
1, young ear selection is got: choose and be in the young fringe that Ying's flower primordium forms the phase, seed forms as yet in the young fringe, and axis is also softer, does not also form basic branch.
2, carry out just for inducing with the initial culture base: it is cut into 2-3 centimetre of segment, use earlier the 75%(volume ratio, below identical) alcohol disinfecting (30-35s), use the 0.1%(mass volume ratio then, below identical) mercury chloride soaks (3-5 minute), use aseptic water washing (3-5 time) again, it is seeded in the first on the inducing culture of young fringe.First generation through (30-35 days) induces, and obtains embryo callus subculture.Condition of culture is 24-28 ℃, illumination every day 14-16 hour (adopting the artificial light source irradiation), and light intensity is 2000LX.The initial culture base is that following prescription selects one: the A:MS medium adds kinetin KT1-5mg/L, methyl NAA0.1-0.5mg/L, sucrose 30g/L, PhytagelR 3g/L or B:MS medium add kinetin KT1-7mg/L, methyl NAA0.1-0.5mg/L, 6 aqueous magnesium chloride MgCl
26H
2O800mg/L, sucrose 30g/L, PhytagelR 3g/L.Adopt any medium in these two kinds of medium can both induce embryo callus in good condition, grow began more at young fringe base portion in 9-11 days, and callus is in good condition, is white or faint yellow loose callus.
3, with giving birth to the seedling medium to giving birth to the seedling successive transfer culture: the embryo callus subculture that is obtained subculture on subculture medium is given birth to seedling, (25-30 days) subculture once, growth coefficient is 5-6.Condition of culture is 24-28 ℃, illumination every day 14-16 hour (adopting the artificial light source irradiation), and light intensity is 2000LX.Giving birth to the seedling culture medium prescription is that following prescription selects one: the A:MS medium adds kinetin KT1-2mg/L, methyl NAA0.1-0.3mg/L, sucrose 30g/L, active carbon 3-8g/L.PhytagelR 3g/L or B:MS medium add kinetin KT1-2mg/L, methyl NAA0.1-0.3mg/L, 6 aqueous magnesium chloride MgCl
26H
2O800mg/L, sucrose 30g/L, active carbon 3-8g/L, PhytagelR 3g/L.Forward after wherein any living seedling medium embryo callus subculture to 2-4 days, it is green that callus begins to change, and began differentiation in 6-8 days to sprout, and can form little seedling about 9-11 days.
4, carry out root induction with the root induction medium: the root induction on the root induction medium of living seedling, cultivate (25-30 days), rooting rate reaches more than 70%.Condition of culture is 24-28 ℃, illumination every day 14-16 hour (adopting the artificial light source irradiation), and light intensity is 2000LX.The root induction medium is that following prescription selects one: A:1/2 MS medium adds kinetin KT2-3mg/L, methyl NAA0.1-0.5mg/L, sucrose 30g/L, PhytagelR 3g/L or B:MS medium add kinetin KT2-3mg/L, methyl NAA0.1-0.3mg/L, sucrose 30g/L, active carbon 3-8g/L, PhytagelR3g/L.Long seedling to 10-15 centimetre is forwarded on any one root media, and seedling will differentiate a large amount of whites or yellow root in general 4-6 days, and well developed root system.
5, domestication is transplanted: in the greenhouse, with robust growth, plant height is transplanted in the domestication nursery of being made up of vermiculite at 10-15 centimetre tissue cultivating seedling and tames.7 timings every morning spray domestication culture fluid and clear water to seedling, approximately behind the 15-20d, look the seedling growing way, can be transplanted in the nursery.Greenhouse experiment is 15 ℃-25 ℃, and halogen light is used in illumination, illumination (14-16 hour), and intensity of illumination is 2000LX.After being transplanted to the nursery, seedling grows fine, robust growth, and well developed root system, survival rate is higher, has obtained the Caulis Miscanthis floriduli seedling of a large amount of stalwartnesses.
This method embryo callus subculture healing rate height, callus are induced living seedling rate height again, and after tissue cultivating seedling domestication was transplanted, the survival rate height was applicable to the industrialization production of Caulis Miscanthis floriduli seedling and lays the foundation for follow-up transgenosis.
The present invention compared with prior art has the following advantages and effect:
A) sterilization method of the present invention is simple, and the efficient height, and is just almost nil for pollution rate.
B) the callus reproduction rate height that obtains of the present invention is about 100%.
C) the present invention has once obtained tissue-culturing rapid propagation and transgenosis group training system, for follow-up transgenosis work lays the first stone.
Description of drawings
Fig. 1 is a kind of Caulis Miscanthis floriduli body embryo callus schematic diagram.
This figure is a Caulis Miscanthis floriduli with young fringe is explant everywhere callus again, and as we can see from the figure, it is faint yellow or white, loosely organized that callus is.
Fig. 2 is a kind of Caulis Miscanthis floriduli seedling schematic diagram.
This figure is that callus changes growth of seedlings figure on the differential medium over to, and visible seedling growing way is better among the figure, and vitality is strong, and has root to grow.
Fig. 3 is that schematic diagram is transplanted in a kind of Caulis Miscanthis floriduli domestication.
This figure is that seedling replanting is in the vermiculite that is added with nutrient solution.
Embodiment
Embodiment 1:
A kind of awns platymiscium Caulis Miscanthis floriduli body embryo tissue culture quick propagation culturing method the steps include:
1, young ear selection is got: choose and be in the young fringe that Ying's flower primordium forms the phase, seed forms as yet in the young fringe, and axis is also softer, does not also form basic branch.
2, carry out just for inducing with the initial culture base: it is cut into 2-3 centimetre of segment,, soaked 5 minutes with 0.1% mercury chloride then, use aseptic water washing again three times, it is seeded in the first on the inducing culture of young fringe earlier with 75% alcohol disinfecting 30s.First generation through 35 days induces, and obtains embryo callus subculture.Condition of culture is 25 ℃, and every day, illumination was 16 hours, and light intensity is 2000LX.Induce the initial culture base to be: the A:MS medium adds kinetin KT1-5mg/L, methyl NAA0.1-0.5mg/L, and sucrose 30g/L,, PhytagelR 3g/L.Growing about 10 days begins more at young fringe base portion, and callus is in good condition, is white or faint yellow loose callus.
3, with giving birth to the seedling medium to giving birth to the seedling successive transfer culture: the embryo callus subculture that is obtained subculture on subculture medium is given birth to seedling, 30 days subcultures once, growth coefficient is 5-6.Condition of culture is 25 ℃, and every day, illumination was 16 hours, and light intensity is 2000LX.Giving birth to the seedling culture medium prescription is: the A:MS medium adds kinetin KT1-2mg/L, methyl NAA0.1-0.3mg/L, sucrose 30g/L, active carbon 3-8g/L, PhytagelR 3g/L.Forwarded embryo callus subculture to wherein any living seedling medium about 3 days afterwards, it is green that callus begins to change, and begins differentiation about 7 days and sprout, and can form little seedling about 10 days.
4, carry out root induction with the root induction medium: living seedling in root induction on the root induction medium, cultivated 30 days, rooting rate reaches more than 70%.Condition of culture is 25 ℃, and every day, illumination was 16 hours, and light intensity is 2000LX.The root induction medium is: A:1/2 MS medium adds kinetin KT2-3mg/L, methyl NAA0.1-0.5mg/L, sucrose 30g/L, PhytagelR 3g/L.Long seedling to 10-15 centimetre is forwarded on the root media, and left and right sides seedling will differentiate a large amount of whites or yellow root in general 5 days, and well developed root system.
Domestication is transplanted: in the greenhouse, with robust growth, plant height is transplanted in the domestication nursery of being made up of vermiculite at 5-6 centimetre tissue cultivating seedling and tames.Regularly spray domestication culture fluid and clear water to seedling every day, approximately behind the 15d, looks the seedling growing way, can be transplanted in the nursery.20 ℃ of greenhouse experiments, halogen light is used in illumination, illumination 16 hours, intensity of illumination is 2000LX.After being transplanted to the nursery, seedling grows fine, robust growth, and well developed root system, survival rate is higher, has obtained the Caulis Miscanthis floriduli seedling of a large amount of stalwartnesses.
Claims (1)
1. an awns platymiscium Caulis Miscanthis floriduli body embryo tissue culture quick propagation culturing method the steps include:
A, young ear selection are got: choose and be in the young fringe that Ying's flower primordium forms the phase;
B, carry out just for inducing with the initial culture base: it is cut into 2-3 centimetre of segment, first alcohol disinfecting 30-35s with 75% volume ratio, mercury chloride with 0.1% mass volume ratio soaked 3-5 minute then, use aseptic water washing 3-5 time again, it is seeded in the first on the inducing culture of young fringe, first generation through 30-35 days induces, obtain embryo callus subculture, condition of culture is 24-28 ℃, illumination every day 14-16, light intensity is 2000LX, and the initial culture base is: the A:MS medium adds kinetin KT1-5mg/L, methyl NAA0.1-0.5mg/L, sucrose 30g/L, PhytagelR 3g/L or B:MS medium add kinetin KT1-7mg/L, methyl NAA0.1-0.5mg/L, 6 aqueous magnesium chloride MgCl
26H
2O800mg/L, sucrose 30g/L, PhytagelR 3g/L adopts any medium in these two kinds of medium can both induce embryo callus, grows to begin more at young fringe base portion in 9-11 days, be white or faint yellow loose callus;
C, with giving birth to the seedling medium to giving birth to the seedling successive transfer culture: the embryo callus subculture that is obtained subculture on subculture medium is given birth to seedling, 25-30 days subcultures once, growth coefficient is 5-6, condition of culture is 24-28 ℃, illumination every day 14-16 hour, light intensity is 2000LX, giving birth to the seedling culture medium prescription is: the A:MS medium adds kinetin KT1-2mg/L, methyl NAA0.1-0.3mg/L, sucrose 30g/L, active carbon 3-8g/L, PhytagelR 3g/L or B:MS medium add kinetin KT1-2mg/L, methyl NAA0.1-0.3mg/L, 6 aqueous magnesium chloride MgCl
26H
2O800mg/L, sucrose 30g/L, active carbon 3-8g/L, PhytagelR 3g/L forwards after wherein any living seedling medium embryo callus subculture to 2-4 days, and it is green that callus begins to change, and began differentiation in 6-8 days to sprout, and formed little seedling in 9-11 days;
D, carry out root induction with the root induction medium: the root induction on the root induction medium of living seedling, cultivated 25-30 days, condition of culture is 24-28 ℃, illumination every day 14-16 hour, light intensity is 2000LX, the root induction medium is: A:1/2 MS medium adds kinetin KT2-3mg/L, methyl NAA0.1-0.5mg/L, sucrose 30g/L, PhytagelR 3g/L or B:MS medium add kinetin KT2-3mg/L, methyl NAA0.1-0.3mg/L, sucrose 30g/L, active carbon 3-8g/L, PhytagelR3g/L, long seedling to 10-15 centimetre forwards on any one root media, and seedling differentiated white or yellow root in 4-6 days;
E, domestication are transplanted: in the greenhouse, with robust growth, plant height is transplanted in the domestication nursery of being made up of vermiculite at 10-15 centimetre tissue cultivating seedling and tames, regularly spray domestication culture fluid and clear water to seedling every day, behind the 15-20d, be transplanted in the nursery, greenhouse experiment is 15 ℃-25 ℃, halogen light is used in illumination, illumination 14-16 hour, intensity of illumination was 2000LX, obtained the Caulis Miscanthis floriduli seedling.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102524068A (en) * | 2011-12-29 | 2012-07-04 | 湖北光芒能源植物有限公司 | Method for quickly reproducing miscanthus plant miscanthus and triarrhena hybrid NO.9 somatic embryo tissue culture |
| CN102577983A (en) * | 2012-03-23 | 2012-07-18 | 湖南农业大学 | Rapid propagation method for Miscanthus floridulus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101889552A (en) * | 2010-08-23 | 2010-11-24 | 湖南农业大学 | Rapid breeding method of miscanthus sinensis |
| CN102148756A (en) * | 2011-01-26 | 2011-08-10 | 武汉邮电科学研究院 | IPv6 over low power wireless personal area network (6LoWPAN) neighbor discovery-based tree routing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101889552A (en) * | 2010-08-23 | 2010-11-24 | 湖南农业大学 | Rapid breeding method of miscanthus sinensis |
| CN102148756A (en) * | 2011-01-26 | 2011-08-10 | 武汉邮电科学研究院 | IPv6 over low power wireless personal area network (6LoWPAN) neighbor discovery-based tree routing method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102524068A (en) * | 2011-12-29 | 2012-07-04 | 湖北光芒能源植物有限公司 | Method for quickly reproducing miscanthus plant miscanthus and triarrhena hybrid NO.9 somatic embryo tissue culture |
| CN102524068B (en) * | 2011-12-29 | 2014-01-01 | 湖北光芒能源植物有限公司 | Method for quickly reproducing miscanthus plant miscanthus and triarrhena hybrid NO.9 somatic embryo by tissue culture |
| CN102577983A (en) * | 2012-03-23 | 2012-07-18 | 湖南农业大学 | Rapid propagation method for Miscanthus floridulus |
| CN102577983B (en) * | 2012-03-23 | 2013-11-06 | 湖南农业大学 | Rapid propagation method for Miscanthus floridulus |
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